Biological signaling cascades frequently involve the covalent modification of proteins. Lysine is a frequent target of such post-translational modifications; in particular, the epsilon-amine of lysine can be substituted with one, two or three methyl groups and can be acetylated. While these modifications have been found in the context of many proteins, particularly noteworthy is the role of lysine post-translational modification in the context of histones, the proteins that serve as the foundation of chromatin. The basic unit of chromatin is the nucleosome, composed of 147 bp of DNA wrapped around an octamer of histones. Lysine methylation and acetylation have been demonstrated to play an important role in the regulation of chromatin structure and thereby are involved in the regulation of processes including transcription, DNA-repair and replication.
Studies in model organisms implicate histone lysine methylation as particularly important for defining the epigenetic status of a cell. The ε-amine of lysine is subject to mono-, di-, or trimethylation. Each methylation state may have a distinct regulatory impact through modulating the binding of different effector proteins (Martin, C. and Zhang, Y., Nat Rev Mol Cell Biol, 6:838-849(2005); Sims, R. J., 3rd et al., Trends Genet, 19:629-639 (2003)). Consistent with this notion, plant homeodomains (PHD) in the NURF remodeling complex and in the tumor suppressor ING2 bind with specificity for trimethylated over dimethylated Lys4 of histone H3 (Li, H. et al., Nature, 442:91-95 (2006); Pena, P. V. et al., Nature, 442:100-103 (2006); Shi, X. et al., Nature, 442:96-99 (2006); Wysocka, J. et al., Nature, 442:86-90 (2006)). The functional consequences of lysine methylation, in addition to being degree dependant, are also determined by the site of methylation (Lachner, M. et al., J Cell Sci, 116:2117-2124 (2003)). For example, while trimethylation at Lys4 is associated with euchromatin and transcriptional activation (Santos-Rosa, H. et al., Nature, 419:407-411 (2002)), trimethylation of H3 Lys9 is a well-established marker of heterochromatin and associated with transcriptional repression (Lee, D. Y. et al., Endocr Rev, 26:147-170 (2005); Rea, S. et al., Nature, 406:593-599 (2000)).
Recent years have seen the identification of numerous enzymes responsible for lysine methylation and demethylation, as well as down-stream effectors that bind to specific methyl lysine residues in histones (Grewal, S. I., and Moazed, D., Science, 301:798-802(2003); Martin, C. and Zhang, Y., Nat Rev Mol Cell Biol, 6:838-849(2005)). For instance, the Lys9-specific methyltransferase SUV39H1 (and its orthologues in other organisms) has been implicated in transcriptional silencing (Ivanova, A. V. et al., Nat Genet, 19:192-195(1998); Rea, S. et al., Nature, 406:593-599 (2000)) and interacts genetically and biochemically with the heterochromatin associated protein HP1α (and its orthologues). Indeed, Lys9 methylation is recognized by the chromodomain of HP1α, which itself recruits SUV39H1 and is believed to oligomerize, causing a repressive chromatin structure by spreading along the chromatin (Grewal, S. I., and Moazed, D., Science, 301:798-802(2003)).
Methyl lysine residues in nucleosomal histones are hypothesized to mediate interactions with the macromolecular complexes that regulate transcription, replication, and DNA-repair. Investigating how lysine modifications influence the activity of these factors would be facilitated by a biochemical system that allows testing of specific methylation patterns on any histone. In particular, nucleosomes reconstituted from homogeneous preparations of recombinant histones, ideally with every possible methylation state at each site of interest, would allow systematic examination of the events downstream of lysine methylation.
Current methods to introduce methylation into recombinant histones include biosynthetic approaches or semi-synthesis. The use of enzymes to methylate lysine residues is limited by the availability of specific methyltransferases. Even in cases where an appropriate methyltransferase is available, these reactions are difficult to drive to completion and can lead to uncontrolled degrees of methylation or heterogeneity with respect to site-specificity.
Semi-synthetic methods to construct modified histones using native chemical ligation have been reported (He, S. et al., Proc Natl Acad Sci USA, 100: 12033-12038 (2003); Shogren-Knaak, M. A. et al., J Biol Chem, 278:15744-15748 (2003); Shogren-Knaak, M. A. and Peterson, C. L., Methods Enzymol, 375:62-76 (2004)). This approach was instrumental in demonstrating a role for H4 Lys 16 acetylation in antagonizing chromatin compaction (Shogren-Knaak, M. et al., Science, 311:844-847 (2006)), underscoring the utility of homogeneously modified histones for investigating the impact of lysine modifications on chromatin function. Nonetheless, the semisynthesis of modified histones is currently limited to modifications at only N-terminal residues (residues 1-30) and requires the synthesis of large quantities of modified peptide thioesters.
Thus, there is a need in the art for improved reagents and methods for the generation of histone proteins with site specific post-translations modifications, particularly methylation or acetylation. One need in the art is an efficient means to reconstitute nucleosomes with site-specific mono, di- and trimethylation at positions throughout the entire sequence of each histone.